Long-distance transport of many organelles inside eukaryotic cells is driven by the dynein and kinesin motors onmicrotubule filaments. More than 30 years since the discovery of these motors, unanswered questions include motor–organelle selectivity, structural determinants of processivity, collective behaviour of motors and track selection within thecomplex cytoskeletal architecture, to name a few. Fluorescence microscopy has been invaluable in addressing some of thesequestions. Here we present a review of some efforts to understand these sub-microscopic machines using fluorescence.